The human system involves the production of serum proteins, known as antibodies, by the lymphoid cell series capable of reacting with antigenic determinants which trigger their production. Since the conventional response of the immune system to an antigen with many antigenic determinants is the production of antibodies to each determinant, the antiserum produced is heterologous in nature and polyclonal, or produced by many different cells each producing antibodies to a specific determinant. Antigenic determinants may be referred to as epitopes when more than one occurs on a single molecule and particularly when each elicits an antibody developing, immune response. A single antibody molecule is specific for a unique antigenic determinant or epitope.
Monoclonal antibodies are uniform antibodies directed to a single determinant or epitope on the antigen molecule which may be repeated at several sites of the molecule. Obviously, to produce such monoclonal antibodies in vitro requires selecting a homogeneous antibody having the desired specifications from numerous antibodies elicited in a conventional polyclonal response. The basic technology for in vitro production of homogeneous, highly specific, monoclonal antibodies was developed by Kohler, G. and Milstein, C. (Nature 256:495-497, 1975) known as hybridoma technique. This method involved the immunizing of mice with antigens resulting in the harvesting of antibody-producing cells from those animals, and fusing these antibody-producing cells with a strain of antibody non-producing myeloma cells, e.g. plasma cell tumor cells, to produce hybridomas. These hybridomas are robust cells which have all of the in vitro survival and growth stamina of the myeloma cell line and antibody producing quality of the B lymphocytes with which it was fused. The hybridomas thus produce monoclonal antibodies and may either be cultured in vitro or may be grown as tumors in a host animal. Since each antibody-producing cell produces a single, unique antibody, the monoclonal cultures of hybridomas each produce a homogeneous antibody which may be obtained either from the culture medium of hybridoma cultures grown in vitro or from the cells, injected into the peritoneal cavity of mice producing ascitic fluid, or serum of a hybridoma tumor bearing host animal.
Although the general scheme of hybridoma and monoclonal antibody production is well known at this stage of implementation, great care must be exercised in the separation and maintenance of hybridoma cells in culture. Isolated clones have been known to produce antibodies against a subject antigen which differs from clone to clone since antibodies produced by different cells may react with different antigenic determinants on the same molecule. Adequate testing of the resulting antibody or antibody-containing medium, serum or ascitic fluid is essential. It is necessary to characterize the antibody of each clone which contributes to the complexity of producing monoclonal antibodies which are to be utilized in both diagnostic and therapeutic applications.
In developing a desired monoclonal antibody, one must identify and locate the antigenic determinant which will elicit a specific antibody to bind with it. Or, conversely, develop several hundred hybridoma clones from fusions performed and exhaustively screen them against normal and non-normal tissue and different antigens in identifying and defining that clone which produces the antibody with desired binding specificity. According to this invention the antibody produced detects structural differences on cell surface markers associated with the onset of adenocarcinoma and squamous cell carcinoma, the primary types of carcinoma. The primary object of this invention is to create and maintain hybridomas which produce monoclonal antibodies which will bind with such a particular antigenic determinant to achieve this desired functional specificity.
It is known that monoclonal antibodies may be labeled with a selected variety of labels for desired selective usages in detection, diagnostic assays or even therapeutic applications. In each case, the binding of the labelled monoclonal antibody to the determinant site of the antigen will signal detection or delivery of a particular therapeutic agent to the antigenic determinant on the non-normal cell. A further object of this invention is to provide the specific monoclonal antibody suitably labelled for achieving such desired selective usages thereof.
This invention has particular application to achieving identification of carcinoma cells which occur in the specific diseases of adenocarcinoma and squamous cell carcinoma, the primary forms of carcinoma.